The most robust and least costly synthetic chemistry for oligonucleotide synthesis is that already well established for conventional use (Caruthers, 1991). The direction of synthesis of the oligonucleotide chains is typically in the 3′- to 5′-direction, with the first monomeric unit covalently attached through its 3′—OH group and a linker to a solid phase such as controlled porosity glass or plastic beads. The method is described as solid phase, but all other reactants are provided by a surrounding liquid phase: the reactions occur on rather than in the solid phase. The monomeric building blocks are nucleosides with an acid labile 5′-O-dimethoxytrityl group (DMT−) protecting the nucleoside 5′—OH position, and a cyanoethyl-phosphoramidite group at the 3′—OH position involving a repetitive sequence of chemical reactions involved in oligonucleotide synthesis. Stepwise addition of each monomeric unit to the growing oligonucleotide requires deprotection of the existing terminal 5′-nucleotide by removal of DMT with added acid prior to coupling with a new nucleoside via a 3′-phosphoramidite group (Caruthers 1991; Beaucage & Iyer, 1992). Acid-labile protecting groups other than DMT may be used (Lehmann et al, 1989) without altering the general principles of the procedure. The direction of synthesis can be altered by switching the substituents at the 3′- and 5′-positions.
Pease et al (1994) created a photodirected method of oligonucleotide synthesis by replacing the DMT-blocking group with a photosensitive one that leaves on illumination. All other reactions were unchanged, but the solid phase was a sheet of glass rather than granules or beads. This photodependent method of oligonucleotide deprotection at the 5′—OH position was used with patterned illumination to create different oligonucleotides at different but spatially addressable locations on a planar surface, i.e. an oligonucleotide array. Because photo-deprotection at the 5′—OH position must be essentially complete to preserve synthetic yields over typically 20-25 synthetic cycles for each oligonucleotide, illumination must be prolonged for 8-10 photolytic half-lives of the photosensitive protecting group. Light sensitivity is therefore low but sensitivity to stray light is not (Garland & Serafinowski, 2002). Expensive reagents, poor light sensitivity and photolithographic masks all increase the cost of array fabrication. Replacement of photolithographic masks by pattern projection with a digital light modulator makes for much improved flexibility (Singh-Gasson et al, 1999). However, the low light sensitivity of direct photodeprotection allied with limits to the UV light intensities that digital light modulators can tolerate constrains this maskless method to serial rather than parallel synthesis, i.e. one full size array (currently 1024×768 pixels) per complete synthesis. The resultant low throughput maintains high cost. The major advantage of the maskless method is in removing the very substantial initial cost of making a new set of 80-100 photolithographic masks for custom-designed arrays. Arrays made using the photodirected chemistry described above are available commercially, but there is a major need amongst users or would-be users for arrays that are less expensive than those currently available.
The fabrication of high density oligonucleotide arrays can also be carried out using the conventional DMT-based chemistry. This variation requires that the acid responsible for removal of DMT-groups is provided in a spatially addressable manner at the planar surface on which oligonucleotides are to be synthesised.
Irrespective of whether the acid is added as such, generated photochemically (Wallraff et al, 1997; Gao et al, 1998, Serafinowski & Garland, 2003) or electrochemically (Southern & Egeland, 2004) some means must be found to restrict diffusion of acid from its point of generation or application to other sites on the array surface, otherwise the spatial separations that define an array will be lost.
A variety of methods have been used to localize acid to discrete areas on a planar surface. Where the acid is photogenerated in solution, surface tension wells (Gao et al, 2001) or physical wells (Zhou et al, 2004) have been successfully used, but the surface density of the resulting arrays was not high, about 104 elements or features per cm2. Low array densities are also obtained using local application of acid solution by mechanical pins, micro-jets, micro-channels or elastomeric templates.
Solid matrices of organic polymers have been used for many years as transparent and inert matrices in which the photochemistry of incorporated light-sensitive molecules under conditions of restricted translational and rotational diffusion could be studied by optical methods (e.g. Cowell & Pitts, 1968). Similar matrices have come into widespread use as films for photolithographic patterning in the fabrication of integrated semiconductor devices, where they are used as photoresists. In this use, a solid film of photoresist composition is created on a planar semiconductor substrate, typically silicon, by spin coating from a solution of the photoresist composed of an organic polymer and other components including a photoacid generator. A heating step (pre-exposure bake) is invariably used to drive off residual solvent remaining from the film casting solution that would otherwise increase photoacid diffusion in the film to levels that might give unacceptable loss of photolithographic resolution. Exposure of the film to patterned light illumination to generate photoacid followed by post-exposure baking alters the solubility of the illuminated areas, such that exposure to a particular solvent or mixture of solvents differentially removes polymer from the illuminated or non-illuminated areas. The resulting polymer pattern then determines access in the exposed areas of the substrate to treatments such as vapor etching or layer deposition that determine the structure of the integrated circuit (Thompson et al, 1994).
Translation of photoacid-dependent photolithographic methods from the semiconductor industry to oligonucleotide array fabrication would at first sight seem attractive. However, the polymer and PAG requirements for photodirected synthesis of oligonucleotide arrays differ in several significant ways from those for manufacture of integrated semiconductor devices, as follows. Firstly, with one exception (Pease et al, 1998) the polymer is required only as a diffusion-restricting solid matrix for photoacid, and not as a photo-patterned resist for determining access of other reagents to the substrate surface as in semiconductor device fabrication. Moreover, in the synthesis of oligonucleotide arrays the molecules being synthesised are easily damaged by strong acids or free radicals, yet these are generated by many of the PAGs widely used in the electronics industry. This damage may be exacerbated by the high temperatures used for post-exposure baking. Finally, for the synthesis of oligonucleotide arrays, the stepwise yield for the photoacid-dependent steps in the photodirected synthesis must be very close to 100%, otherwise the overall yield of desired product will fall with each successive synthetic cycle, to be replaced by products of incorrect sequence. A similar quantitative yield is not demanded of the chemistry involved in photolithography.
Despite these potential hurdles photolithographic generation of acid in solid diffusion-restricting films of organic polymers would avoid the problems posed by diffusion of acid in liquid solution, and offer 100-fold or greater increases in array density. The high array densities combined with lower reagent costs and industrial-scale fabrication offer the prospect of substantial reductions in the fabrication cost of oligonucleotide arrays, resulting in their much wider availability to users in biomedical research and its applications. However, getting the detritylation step chemistry to work properly and achieve >99% deprotection of the oligonucleotide-5′—OH group in a diffusion restricting polymer has been problematical, and this approach has not at present been satisfactorily implemented for oligonucleotide array fabrication.
Part of this difficulty arises from reversibility of acid-dependent removal of protecting groups such as DMT. In conventional solid phase syntheses of peptides or oligonucleotides the released acid-labile protecting group can be washed away with deprotection solution from the site of production at the solid phase, enabling the deprotection reaction to proceed to completion. Washing the solid phase with the deprotecting solution is simple and inexpensive. Nevertheless alternative strategies have been proposed and exemplified, even if not widely used. They depend on providing in the (typically acid) deprotection solution a compound that acts as a scavenger for the protecting group released at the surface of the solid phase.
An early example of the use of a scavenging agent was given by Brenner et al (1984) and Davison et al (1987) for the use of triethylsilane as a carbocation scavenger in the acidic deprotection of a tritylated thiol derivative. Reese et al (1986) used pyrrole to react irreversibly with DMT carbocation in solution phase oligonucleotide synthesis. Mehta et al (1992) used triethylsilane to react irreversibly with the carbocations released by acid from peptides protected by t-butyl or t-butoxycarbonyl groups. Ravikumar et al (1996) described the use of a variety of scavengers in acid solution used for deprotection of oligonucleotide OH-groups protected by DMT or related groups that also convert to carbocations when exposed to acid. The scavengers included anisole, thioanisole, benzyl mercaptan, ethanediol, pyrrole and silanes of general formula R3SiH where R is C1-C4-alkyl, phenyl, or phenyl mono-substituted by halo, nitro- or C1-C4-alkyl. Other possible carbocation scavengers are indole, thiophen and furan, either substituted or non-substituted; phenol, resorcinol, 1,3 dimethoxy benzene, 1,3,5-trimethoxybenzene, and dimethylsulfide.
In the past 10 years, there have been six approaches to the use of photoacid dependent methods in the fabrication of oligonucleotide arrays. One of them, using photoacid in solution, appears to have been technically successful but required sophisticated and expensive microfluidic devices to define the physical boundaries of each array element, which in any case were relatively large and gave low density arrays (Gao et al, 2004. Xu et al, 2004. ). The other five approaches have used photolithographic methods applied to diffusion-restricting solid films at the array surface.
In a first such approach, Wallraff et al (1997) used poly(methylmethacrylate) (“PMMA”) as a solid matrix containing one of a variety of PAGs and applied it to glass surfaces on which oligonucleotide arrays were to be synthesised. The sequence of reactions was as follows:
First, a photoacid generator on illumination produces acid (Equation 1) which then dissociates to anion and proton (Equation 2). The extent of dissociation depends on the pKa of the acid:PAG+light=Residual photoproduct(s)+photoacid  (1)Photoacid=Anion−+H+  (2)
“Residual photoproducts” covers those photoproducts other than the photoacid itself: for example, the substituted 2-nitroso-compounds remaining after photolysis of substituted 2-nitrobenzyl esters. Generated protons participate as a substrate for the detritylation of dimethoxytrityl-blocked oligonucleotides (Equation 3), not as a catalyst. The products of the reaction are the oligonucleotides with an unblocked 5′- or 3′—OH group, depending on the chosen direction of synthesis, plus the dimethoxytrityl cation.DMT-oligonucleotide+H+=DMT++HO-oligonucleotide  (3)
When the environment is solid and aprotic Equation (3) may be more appropriately written with direct participation of the photoacid HA and the formation of ion pairs rather than free ions, as in Equation (4):DMT-oligonucleotide+HA=DMT+.A−+HO-oligonucleotide  (4)
Under the conditions used by Wallraff et al (1997) generation of a preferred carboxylic photoacid failed to achieve adequate detritylation, even if heated to 90-100° C. for 2-3 min. The use of much stronger acids, also with heating, did achieve full detritylation, but caused unacceptable depurination.
In a second approach, conventional photoresists were used to determine access of either added 20% dichloroacetic acid solution (McGall et al, 1996) or trichloroacetic acid vapor (Pease et al, 1998) to 5′-DMT-O-oligonucleotides attached to the underlying substrate, which in all cases was glass. For technical reasons, a layer of polymer was required under the layer of photoresist, thereby involving two spin-coatings and their associated pre- and post-illumination baking steps. The stepwise synthetic yield was 90% and therefore not usable.
In a third approach, Beecher et al (1997, 2000) turned to a chemical amplification method to overcome the inadequate detritylation obtained by Wallraff et al (1997) with weaker acids such as haloacetic or alkylsulphonic acids. In the amplification method, photoacid generated as in Equation (1) catalyzes at elevated temperature the thermolysis of a compound known as an enhancer. For example, 1,4-cyclohex-2-enediylbis-(pentafluorobenzoate) when exposed to both acid and heat decomposes as shown below (Equation 5) to give a net gain of two molecules of acid (pentafluorobenzoic) for each molecule of enhancer that undergoes thermolysis. The photoacid is not the detritylating acid; it acts as a catalyst to initiate the thermolytic and acid-producing decay of the enhancer.

In a fourth approach, Serafinowski & Garland (2003, 2004) described a series of substituted 2-nitrobenzyl esters that, when photolysed, release by an intramolecular rearrangement an acid (trichloroacetic) that is capable of effecting the detritylation step of oligonucleotide without generating potentially damaging photochemical products. Two such esters were tested in a photo-dependent variation of otherwise conventional solid-phase oligonucleotide synthesis on controlled porosity glass. Synthetic yields of 99-100% were obtained (Serafinowski & Garland, 2003). Some of these esters form solid, optically clear films, and when formed as photosensitive films in contact with an underlying planar glass surface carrying covalently attached DMT-T can achieve >98% detritylation on illumination. Post-exposure heating is not required. These same esters are also effective when incorporated in a polymer film, provided that the polymer lacks electronegative heteroatoms that may otherwise weaken the acid by hydrogen bonding (Garland & Serafinowski, 2006).
In a fifth approach Goldberg et al (2005) used as the photoacid generator either 2,6-dinitrobenzyltosylate or a substituted diphenyliodonium hexafluorophosphate in PMMA films on glass. This approach differed from that of Wallraff et al (1997) in two ways: the post-exposure baking step was omitted and a base was incorporated in the film to prevent inappropriate acidification of the films by the relatively strong photoacids. Fidelity of oligonucleotide synthesis using 2,6-dinitrobenzyl-tosylate as the photoacid generator was judged by an indirect method to be comparable with the 97-99% achievable with conventional oligonucleotide synthesis using solution phase. The stepwise synthetic yield using the diphenyliodonium hexafluorophosphate was an unsatisfactory 93% as calculated from the 64% overall synthetic yield of a hexamer.
Of the methods described above for oligonucleotide synthesis using patterned photoacid generation in a diffusion-restricting solid, that using photoacid generators based on substituted 2-nitrobenzyl esters of trichloroacetic acid in films of non-hydrogen bonding polymers has a number of advantages. Trichloroacetic acid itself is well tolerated by purine nucleotides, and has been the acid of choice for oligonucleotide synthesis over many years. The photolysis of substituted 2-nitrobenzyl esters proceeds via an intramolecular rearrangement; hydrogen abstraction from other molecules is not required, and potentially aggressive photochemical products are not formed. Methyl- or phenyl-substitution of the α-carbon atom can be used to improve quantum yield without causing instability of the trichloroacetate esters; this is not the case with tosylic esters, which undergo spontaneous intramolecular rearrangement with loss of acid in response to substitution with methyl or phenyl groups at the α-carbon atom.
Other substitutions of the 2-nitrobenzyl structure can be made to give a wide range of photochemical properties and excitation wavelengths. Post exposure heating is not required. Substituted 2-nitrobenzyl-trichloroacetate esters are film forming in their own right, but can be conveniently used in films of polymers that lack electronegative heteroatoms and cannot weaken carboxylic acids by hydrogen bonding to their undissociated carboxyl groups. (Garland & Serafinowski, 2006).
WO 03/000644 (Serafinowski & Garland) provides a way of overcoming some of the drawbacks of the first approach. It discloses a series of substituted 2-nitrobenzyl esters that, when photolysed, release acid that is capable of removing a terminal protecting group from an oligonucleotide being synthesised in an array. These esters are typically used in the first method described above in which the photogenerated acid is capable of directly deprotecting a dimethoxytrityl-blocked oligonucleotide, rather than needing to employ the third method in which photogenerated acid is not involved in the deprotection reaction and instead serves to catalyze a thermolytic reaction in which the deprotecting acid is generated from an enhancer. The incorporation of the substituted 2-nitrobenzyl esters in polymeric films is disclosed. The application also discloses a method of ameliorating the effect of stray light irradiating elements of the array not being designated for synthesis by including small amounts of a buffer at the elements. The buffer serves to neutralize small amounts of acid produced by stray light while not substantially affecting the larger amounts of acid produced at sites designated for oligonucleotide synthesis.
US Patent Application 2004/0110133 follows on from U.S. Pat. No. 6,083,697 (Beecher et al, 2000) and lists polymers that might be used to provide a solid matrix for reactions (1) to (4) described above. The polymers listed were poly(methylmethacrylate), poly(acrylate), poly(ethylenepropylene), polyethylene and polyvinyl chloride. Apart from poly(methylmethacrylate, no exemplification of the use of these polymers was provided.